AtPCaP1 | Arabidopsis thaliana plasma membrane cation-binding protein-1
AS10 931 | Clonality: Polyclonal | Host: Rabbit | Reactivity: A. thaliana, R. sativus, B.rapa, B.oleracea
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It is confirmed that the nock-out mutant (SALK_022955, Col-0 background) has no mRNA or protein of PCaP1. In the immunoblot, crude membrane fractions prepared from wild-type and pcap1 knock-out mutant plants were examined. Therefore the antibody to PCaP1 specifically recognizes PCaP1 and can be used for immunoblotting. For application in immunocytochemistry cross reacting band in proximity of Rubisco has to be removed by incubation of an antibody with a part of memrane with background band.
Protocol for preparation of crude membranes
(1) Homogenize roots or root tips with the buffer without SDS.
(2) Centrifuge the homogenate in Eppendorf tubes at 2 000 rpm for 2 min to remove cell wall, nuclei, plastids and mitochondria.
(3) Centrifuge the obtained supernatant at 30 000 g for 20 min to separate soluble components. Please use adequate tubes for ultracentrifugation.
(4) Suspend the obtained precipitate (crude membranes) with SDS buffer with DTT or beta-mercaptoethanol.
(5) Heat the sample solution obtained the step (4) at 70 C for 5 min.
(6) Apply the denatured sample to SDS-PAGE.
Courtesy Prof. Msayoshi Maeshima, Nagoya University, Japan
AtPCaP1 is a protein associated with plasma membrane with ability to bind Ca, calmodulin and phosphatidylinositol phoshphates. Alternative names: At4g20260, endomembrane-associated protein.
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